The HIV-1 Gag proteins are synthesized as a polyprotein precursor, Pr55Gag, that is cleaved by protease (PR) during virus release from the infected cell. PR-mediated Pr55Gag processing follows a sequence of events that is kinetically controlled by the rate of processing at individual cleavage sites. Completion of the Gag processing cascade is essential for virus maturation and infectivity. In collaboration with Panacos Pharmaceuticals, we found that 3-0-(3-3-dimethyl-succinyl) betulinic acid (PA-457 or bevirimat) potently inhibits HIV-1 maturation by specifically blocking a late step in the Gag processing pathway. We have performed initial characterization of the mechanism of action of PA-457 and have isolated and characterized a number of PA-457-resistant HIV-1 variants. In ongoing and future studies, we will fully define the mechanism of action of PA-457 and in collaboration with Panacos will develop second-generation maturation inhibitors that may be active against both wild-type and PA-457-resistant HIV-1 isolates. [Corresponds to Freed Project 4 in the April 2007 site visit report of the HIV Drug Resistance Program]